Three nitroheterocyclic drugs used to treat humans have worrisome properties. Metronidazole and niridazole are animal carcinogens and are mutagenic to the Ames tester strains. Nitrofurantoin is structurally related to known carcinogens and is also mutagenic. Metabolic reduction of metronidazole and nitrofurantoin has been shown to be obligatory for mutagenesis and for their interaction with macromolecules such as DNA. Our previous work has shown that rat cecal contents can reduce metronidazole and that variety of products are formed. One of these, N-(2-hydroxyethyl) -oxamic acid, has already been characterized. It appears in the urine of conventional but not of germfree rats and thus seems to be formed exclusively by the intestinal microflora. It is proposed to characterize the remaining products of metronidazole reduction by the flora and to determine the role of the flora in their formation by continuing to compare metronidazole metabolism in germfree and conventional rats. In addition it is planned to study the metabolism of nitrofurantoin and niridazole in rat cecal contents and to compare the metabolism of these drugs in germfree and conventional rats to determine the role of the flora in their in vivo metabolism. It is also planned to modify the use of host mediated assays previously described in conventional mice and in gnotobiotic rats to determine whether reductive reactions of the flora on these three drugs are responsible for the formation of reactive mutagens which can be detected in the mouse peritoneum or the rat gastrointestinal tract. In these studies use will be made of reductaseless Ames mutants which only revert with metronidazole in the presence of nitroreductase from an external source such as the flora. It is also planned to determine whether metabolites derived from radioactive metronidazole are different in the tissues of germfree and conventional animals.